A combination of Zinc solubilizing bacteria and ZnO nanoparticles alleviates the toxicity of Ag and TiO2 nanoparticles in Mung bean

doi:10.21203/rs.3.rs-3558916/v1

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A combination of Zinc solubilizing bacteria and ZnO nanoparticles alleviates the toxicity of Ag and TiO₂ nanoparticles in Mung bean

https://doi.org/10.21203/rs.3.rs-3558916/v1

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Zinc-deficiency, a global health challenge affects one-third of the world population. Zinc solubilizing bacteria (ZSB) increases plant vegetative growth, grain’s Zn-content ensuring improved dietary Zn-supply offering an efficient and cost-effective solution. This study aimed at understanding that how the presence of two soil abundant nanoparticles silver and titanium dioxide in the rhizosphere influences the activity of ZSB and plant’s vegetative growth. Two ZSB strains D-7 and D-117 with good zinc solubilization efficiency of 254 and 260%, respectively were used with ZnO-NPs for Zn fortification of Mung bean in the presence of various doses of TiO₂ and Ag-NPs. Two sets of pots, were used, one containing ZSB, Mung bean seeds, ZnO-NPs (20 mg kg^− 1 soil) and various doses of nanoparticles. ZSB-free pots were used as control. The plants were studied for change in rate of seed germination, vegetative growth (shoot and root length, fresh and dry weight), photosynthetic pigment and Zn-content. Lower dose of nanomaterials (50 and 100 mg kg^− 1 soil) slightly promoted the plant growth compared to control. While, higher doses (200 and 400 mg kg^− 1 soil) inhibited the growth. A maximum decrease of shoot length, root length, fresh-weight, and dry-weight of 37.5, 62.3, 57.1, and 53.2%, respectively was observed with 400 mg kg^− 1 soil of Ag-NPs. In the presence of ZSB the decrease was 34.8, 21.3, 41.6, and 37.9, respectively with the same concentration of Ag-NPs. Results presented in this study demonstrate that ZSB alleviate the toxicity of the nanoparticles and improves Zn content in Mung bean.

Biofortification

Zn deficiency

Zn Solubilization

PGPR

Mung bean

Metals being the cofactors and components of various proteins are required by all living organisms including Humans. Micronutrient malnutrition including that of metals in plants is a major obstacle in achieving the goal of food security and improving nutrition’ by 2030, as envisioned by United Nations (Haddad et al., 2016; Poppy et al., 2022). Micronutrient deficiency affects at least two billion people across the globe with varying health and growth consequences (Khan et al., 2022b). Among metals Zinc (Zn) being a component of all the six classes of enzymes is involved in almost all the physiological reactions in all forms of life (Khan et al., 2022b). Deficiency of Zn in soil or its availability in insoluble forms causes Zn deficiency in plants the primary producers of the food chain consequently leading to its malnutrition in higher trophic levels like animals and Humans. Soils that contains DTPA-extractable Zn lower than 0.5 mg kg⁻¹ are commonly considered as Zn deficient (Alloway, 2008). Nearly 50% of the soils used for cereal cultivation contain very little Zn often in a form which is not available to plants (Alloway, 2008; Khan et al., 2022b). Zn solubilizing heterotrophic bacteria solubilize these insoluble forms of Zn present in soil through production of organic acids and siderophores making it available to plants. The bioavailability of Zn also depends on various edaphic factors most important being the soil pH. Zn solubilization bacteria have been tested as inoculum for improving plant Zn content in agronomic studies (Kamran et al., 2017; Costerousse et al., 2018; Saleem and Khan, 2022). These strains regulate soil pH through production of organic acids and are especially helpful in alkaline soil. Lowering of soil pH not only improves the Zn supply, but also affect plant and soil health.

The use of nanomaterials has increased sharply over the years consequently increasing their release in various environment including soil (Keller et al., 2013; Khan et al., 2016; Khan and Malik, 2019). In agriculture sector also the direct use of engineered nanomaterials to improve agricultural productivity has increased. In addition to their use to promote agricultural productivity various unwanted engineered nanomaterials are released in soil affecting plant health agriculture productivity and soil ecosystem (Piccinno et al., 2012; Khan et al., 2022a). Metric tons of Titanium dioxide, Zinc oxide, Ferrous/Ferric oxides, Aluminum oxide and Silver are released every year into the soil affecting soil health and agricultural productivity (Keller et al., 2013; Khan et al., 2022a). These nanomaterials are not only affect plant health but also significantly alter the microbiomes associated with soil and plant. Soil and plant microbiomes are crucial to plant health and influence agricultural productivity in many ways (Berendsen et al., 2012; Khan, 2022). Studies have demonstrated the toxicity of these nanomaterials on plant health and the possible mechanisms of toxicity includes, DNA damage, excessive generation of ROS, inhibition of enzymes and other (Khan et al., 2022a; Kang et al., 2023). In most of the studies the toxicity was observed at higher dose of the nanomaterial and varies with type of nanomaterial, shape, size, and other properties of nanomaterials (Khan, 2020; Kang et al., 2023). On the contrary the growth promoting activities of nanomaterials especially the micronutrient like ZnO, Manganese, and Iron is also reported at low doses (Saleem and Khan, 2022; Ahmed et al., 2023; Salehi et al., 2023). This growth supporting activity can be due to the direct provision of nutrient or due to the change in the activities of associated microbes (Khan et al., 2018). As we earlier reported that the sublethal dose of ZnO nanoparticles induces the production of pyrrolnitrin by Pseudomonas protegens CHAO, resulting in its increased antifungal activity which helps in protecting plants from fungal pathogens (Khan et al., 2018). We reported earlier that ZnO nanoparticles improve photosynthesis in tomato plants and improves chlorophyll content in wheat plants (Faizan et al., 2018; Saleem and Khan, 2022). Very few in-vivo studies have demonstrated as to how soil abundant nanomaterials influence the activity of plant growth promoting bacteria. Especially the influence of these nanomaterials on the activity of Zn solubilizing bacteria in-vivo is not reported.

This study was therefore aimed at understanding the influence of various doses of soil abundant nanomaterial (TiO₂ and Ag) on the activity of ZSB and their consequent toxicity to Mung bean plant in the presence of ZnO nanoparticles as a source of Zn.

To understand the influence of soil abundant nanomaterials on the activity of Zn solubilizing bacteria in the rhizosphere of Mung bean, ZSB bacteria were isolated from the soil. Strains with good Zn solubilization efficiency were used for the study. Commercially available nanoparticles were used for the study as detailed below.

2.1 Characterization of nanoparticles

Nanoparticles of Ag, TiO₂, and ZnO were purchased from Sisco Research Laboratories (SRL, Mumbai, India). The crystalline nature, shape, and size, of the nanoparticles were confirmed using transmission electron microscopy (TEM) and X-ray diffraction (XRD). An X-ray diffractometer (X'Pert, PANalytical, Spectris plc, England) equipped with a Ni filter which uses a Cu-Kα (λ = 1.54056˚A) radiations was used to obtain XRD pattern. For electron microscopy, a well-dispersed suspension of dried powder was prepared in deionized water (10 mg mL^− 1) by sonication for 15 min at 40 W. The suspension was mounted on a carbon-coated copper grid and was airdried before observation. The grid was observed under a JEOL transmission electron microscope (JEOL-2100, JEOL, Tokyo, Japan) at a magnification of 80,000× with an accelerating voltage of 200 kV.

2.2 Soil sampling and isolation of aerobic heterotrophs

Soil samples were collected from sites receiving industrial effluents containing Ni, Zn or untreated sewage as detailed in supplementary table 1. Samples were collected using auger in sterile 50 ml Falcon tube and were labelled immediately. The collected samples were transported at room temperature to the lab and were placed on a dry cool place to avoid moisture accumulation or excessive drying. For the isolation of ZSB, appropriately diluted samples were spread on different media (Nutrient agar, Jensen’s medium, Kenknight and Munaier’s Agar, Pikovskaya’s Agar, and Yeast Mannitol Agar) and the plates were incubated at 28°C for 24–36 h. Colonies appearing on agar plates were picked up and were purified using repeated streaking. Pure cultures were preserved at − 80°C in 30% glycerol and were also maintained as slants at 4 ℃.

2.3 Zinc Solubilization assay

The strains isolated from soil were screened for their ability to solubilize Zn on minimal agar amended with 0.1% insoluble zinc (ZnO and ZnCO₃). An aliquot of 5 µL from the actively growing bacterial culture having an absorbance of 0.5 at 660 nm was spotted on the minimal medium containing zinc. The plates were covered with aluminum foil and incubated in dark at 28 ± 2°C for 14 days. Strains producing clear zone or halo around the colony were selected and diameter of the zone of solubilization was recorded. The Zn solubilization efficiency (ZSE) of the ZSB strains was calculated in percentage (%) by using the following formula.

$$ZSE \left(\%\right)=\frac{\text{D}\text{i}\text{a}\text{m}\text{e}\text{t}\text{e}\text{r} \text{o}\text{f} \text{s}\text{o}\text{l}\text{u}\text{b}\text{i}\text{l}\text{i}\text{z}\text{a}\text{t}\text{i}\text{o}\text{n} \text{h}\text{a}\text{l}\text{o} \text{z}\text{o}\text{n}\text{e}}{\text{D}\text{i}\text{a}\text{m}\text{e}\text{t}\text{e}\text{r} \text{o}\text{f} \text{t}\text{h}\text{e} \text{c}\text{o}\text{l}\text{o}\text{n}\text{y}}\times 100$$

The quantitative assessment of Zn solubilization potential of selected ZSB was determined in liquid minimal broth with ZnO as the insoluble Zn source as detailed by Fasim et al. (Fasim et al., 2002). Briefly 500 µl aliquot of overnight grown ZSB inoculum was added to 50 ml of ZnO supplemented basal liquid medium and the medium was incubated on an orbital shaker at 130 rpm and 28 ± 2 ℃ for 10 days. Uninoculated media was taken as control. After incubation, the broth was centrifuged at 6,100 ×g for 10 min. The supernatant was filtered through Whatman filter paper (No. 42) and the concentration of soluble Zn in the filtered sample was checked using Atomic Absorption Spectrophotometer (GBC 932 Plus, GBC Scientific Equipment Ltd. Melbourne, Australia). The pH of the sample was also monitored using a pH meter.

2.4 In vitro screening for PGP traits

ZSB strains showing good Zn solubilization efficiency were screened for various PGP traits using standard protocols including the production of siderophore (Schwyn and Neilands, 1987), IAA (indole-3-acetic acid) (Patten and Glick, 1996), HCN (Hydrogen cyanide) (Donate-Correa et al., 2005), and Ammonia (Cappuccino et al., 1992). The strains were also tested for their ability to solubilize tricalcium phosphate (Nautiyal, 1999). Briefly, phosphate solubilization was tested on Pikovskaya’s agar medium (Nautiyal, 1999). Production of IAA was checked using Salkowski’s colorimetric technique (Gordon and Weber, 1951), and production of ammonia was checked by using Nessler’s reagent (Cappuccino et al., 1992). Chrome Azurol S agar plates were used to check the production of Siderophore (Schwyn and Neilands, 1987). The production of hydrogen cyanide (HCN) was checked on glycine supplemented King’s B agar (Lorck, 1948). Production of amylase, cellulase and pectinase was checked using standard protocols (Hols et al., 1994; Cappuccino and Sherman, 1999).

2.5 Polyphasic characterization of ZSB

Biochemical tests were performed and sequence of 16S rRNA gene was determined for the polyphasic characterization of efficient ZSB selected for the study. The gene for 16S rRNA was amplified using a set of universal primers (27f: 5′ -AGA GTT TGA TCC TGG CTC AG-3′ and 1492r: 5′-GGT TAC CTT GTT ACG ACT T-3′) and the genomic DNA template extracted from the strains as detailed elsewhere (Khan et al., 2007). The partial 16S rRNA gene sequence of the amplicon was determined using an ABI PRISM 3130 XL Genetic Analyzer (Applied Biosystems, MA, USA). Sequence data was assembled using online Cap3 program and the assembled sequences were compared with the sequences available in the databanks using nBLAST tool (Altschul et al., 1997; Huang and Madan, 1999). The sequences obtained in this study were submitted to the NCBI with accession number OR497752 for D117 and OR497753 for D7. To confirm the phylogenetic position of the isolated strains, the biochemical properties of the strains were determined and compared to the properties of their closest phylogenetic neighbour using the standard protocols (Barrow and Feltham, 1993).

2.6 Activity of ZSB in an environment of Ag and TiO₂ nanoparticles and Mung bean rhizosphere

To understand the influence of Ag and TiO₂ nanoparticles on the activity of ZSB two sets of pots were used (Supplementary table 2). In one set of pots the cultures of two effective ZSB strains and ZnO NPs (20 mg Kg^− 1) as a source of Zn were added, followed by the Mung bean (Vigna radiata) seeds. Various doses (50, 100, 200, and 400 mg kg^− 1) of Ag and TiO₂ NPs were also added to these pots. In the second set of pots the ZSB were not added. Pots were maintained at room temperature (25 ± 3°C) and at a relative humidity in the range of 40–85%. Recommended dose of NPK (urea 100 mg N kg^− 1, KH₂PO₄ 75 mg kg^− 1, and KH₂PO₄ and K₂SO₄125 mg K kg^− 1) fertilizer was added to the pots. The soil was irrigated with tap water at slightly above the field capacity. All the pots were maintained in triplicates and details of various treatments used for the study is given in supplementary table 2.

The rate of germination and various plant growth parameters including root length, shoot length, fresh weight and dry weight were recorded 30 days after sowing. Photosynthetic pigments (Chl a, Chl b and carotenoids) in the leaves were extracted using acetone (Lichtenthaler and Wellburn, 1983). The leaves were crushed using mortar and pestle and were extracted with 80% acetone at 4°C. The extracted samples were filtered and absorbance of the extracts was measured by a UV-Vis spectrophotometer at wavelengths of 663.2, 646.8 and 470.0 nm. The concentration of Chl a, Chl b, and total chlorophyll was calculated using the equation of Arnon (Arnon, 1949).

2.8 Zn concentration in soil and plant tissues

The DTPA extraction method was used to determine the concentration of bioavailable Zn in soil (Lindsay and Norvell, 1978). Briefly, the soil was extracted by mixing the soil and DTPA extraction solution (5 × 10^− 3 M diethylenetriaminepentaacetic acid, 0.01 M CaC1₂ and 0.1 M triethanolamine, pH 7.3) in a ratio of 20:10 (v:w) followed by shaking for 2 h. The total Zn concentration in the leaf tissues was determined by acidic digestion with a mixture of nitric acid, sulfuric acid, and perchloric acid in a ratio of 9:2:1 vol vol^− 1 (Rathje, 1959). A mixture of plant tissue with the acid solution was incubated on a hot plate at 100–120°C till the mixture became colorless. The digested samples were filtered using Whatman filter paper 42 (Whatman, Sigma). The Zn concentrations in the extracted sample was determined using flame furnace atomic absorption spectrometry (GBC Scientific Equipment Ltd. Melbourne, Australia). All samples were extracted and analyzed in triplicate using each of the procedures.

3.1 Characterization of nanoparticles

The shape and size of NPs used in the study was determined using transmission electron microscopy. Representative TEM images of Ag, TiO₂ and ZnO NPs are shown in Fig. 1A-C. The morphology of the Ag NPs is predominated by slightly agglomerated, spherical NPs, with a size range of 20–50 nm and an average size of ~ 32 nm (Fig. 1A). The TiO₂ demonstrate, slightly agglomerated, well-defined spherical morphology, the nanoparticles are in the size range of 15–30 nm with an average diameter of ~ 23 nm (Fig. 1B). The ZnO NPs exhibit highly agglomerated spherical shape morphology where the nanoparticles were in the size range of 10–30 nm with an average size of ~ 20 nm (Fig. 1C). The phase purity and crystallinity of these nanoparticles was confirmed by XRD analysis as shown in Fig. 1D-F. The XRD diffractogram in Fig. 1D point to a face-centered cubic structure of the Ag NPs (JCPDS, file no. 04-0783). Ag shows four distinct reflections at 38.04° (111), 44.50° (200), 64.50° (220), 76.89° (311). For TiO₂, the diffraction peaks are observed at 25.3°, 37.8°, 48.0°, 55.1°, and 62.7° which are corresponding to (101), (004), (200), (105), and (211) crystal planes, respectively (Fig. 1E). This clearly confirms the anatase phases of the TiO₂ NPs corresponding the JCPDS 21-1272 31. The X-ray diffraction pattern of ZnO NPs shows peaks at 2θ = 32.06°, 34.66°, 36.54°, 47.82°, 56.89°, and 63.16°, which are assigned to (100), (102), (101), (102), (110) and (103), respectively (Fig. 1F), indicating a typical polycrystalline wurtzite structure of Zincite, (JCPDS 5-0664).

3.2 Isolation of bacteria screening for Zn solubilization potential and PGP traits

A total of 136 bacterial strains were isolated from five soil samples collected from different locations of district Aligarh, U.P. India (Supplementary table 1). The strains were selected based on different colony morphology from different Agar Medium. The pure culture of the strains was obtained through repeated streaking on nutrient agar plates. These strains were screened for their ability to solubilize Zinc and for the presence of multiple PGP traits. The qualitative Zn solubilization was checked on minimal medium containing insoluble Zn. Out of 136 isolates, forty-six isolates were found to solubilize Zn to various degree (Supplementary table 3). Sixteen strains were considered as good Zn solubilizer with a Zn solubilization zone diameter of > 27 mm on minimal media containing insoluble Zn and a zinc solubilization efficiency (ZSE) of > 245% (Fig. 2 and table 1). Two ZSB strains, namely D-7 and D-117, were selected for final field study based on their Zn solubilization efficiency and multiple PGP traits. Strain D-7 and D-117, show a solubilization zone diameter of 30 ± 0.14 mm and 28 ± 0.18 mm, respectively on minimal medium amended with ZnO. The diameter of Zn solubilization zone, by strain D-7 and D-117, on minimal medium amended with ZnCO₃ was 28 ± 0.19 mm and 26 ± 0.19 mm, respectively. The ZSE of strain D-7 on minimal medium with ZnO was 272.73% and on minimal media containing ZnCO₃ it was 254.55%. The strain D-117 shows a ZSE of 280% and 260% on minimal medium containing ZnO or ZnCO₃, respectively. The decrease in pH probably due to the production of organic acids was checked and it was observed that after 10 days of growth, D-7 strain decreased the pH from 7.4 ± 0.61 to 4.47 ± 1.21, and D-117 decrease the pH from 7.4 ± 0.61 to 5.63 ± 1.42. When screened for Plant Growth Promoting (PGP) traits, strains D-7 and D-117 were found to exhibit multiple PGP traits including siderophore production which helps in Zn solubilization making it available to plants (supplementary table 4).

3.3 Polyphasic characterization of efficient ZSB strains

The strains D-7 and D-117 were subjected to polyphasic characterization for their putative identification. The partial 16S rRNA gene sequences of strain D-7, and D-117 shows a maximum similarity of 99 and 98.3% with Bacillus paramycoides strain OFAh (MN519566), and Pseudomonas aeruginosa strain 2019CK-00034 (CP107257), respectively. Neighbour-joining phylogenetic trees based on partial 16S rRNA gene sequence show that the strains D-7 and D-117 group in a monophyletic clade with Bacillus sp. and P. aeruginosa, respectively (Fig. 3). In addition to sharing 16S rRNA gene sequence similarities, strains D-7 and D-117 share many biochemical traits with their phylogenetic neighbour, including hydrolysis of macromolecules (Gelatin, Casein, and starch), utilization of citrate, indole production and fermentation of various sugars (Supplementary table 5). These biochemical tests further confirm that strains D-7 and D-117 belong to Bacillus sp. and P. aeruginosa, respectively.

3.4 Activity of ZSB in an environment of Ag and TiO₂ NPs in Mung bean rhizosphere

The aim of this study was to investigate how the presence of different doses of Ag and TiO₂ NPs affect the activity of ZSB in the rhizosphere of chickpea. And how the vegetative growth of mung bean and its Zn absorption in terms of tissue Zn content is affected? Before conducting the detailed study, the bioavailability of Zn in the soil used for the study was checked and the soil was found to be deficient in Zn having a DTPA extracted Zn of 0.51 mg kg^− 1. It has also been investigated in an unpublished preliminary study as well as in our previous study that whether bulk ZnO or nano ZnO was a better source of Zn. ZnO NPs were confirmed to illicit better growth response and to significantly improve Zn content in plants compared with the same dose of bulk ZnO (Saleem et al., 2023a).

To evaluate the influence of Ag and TiO₂ NPs on the plant growth promoting activity of ZSB, strains Bacillus sp. D-7 and P. aeruginosa D-117 along with ZnO NPs were added to the soil as source of Zn. Various doses of Ag and TiO₂ NPs were added to the soil and finally Mung bean seeds were sown in the soil. While, a similar set of pots without the ZSB was used for comparison. Rate of germination and plant growth parameters were checked following the treatment with various doses of Ag and TiO₂-NPs.

3.4.1 Change in rate of germination: It was observed that the germination rate of the Mung bean seeds changed with different doses of TiO₂ and Ag (Table 2). Compared to control an increase of 7% in germination rate was observed when ZSB strains were added to the soil. An increase of 25% was observed when in addition to ZSB, bulk ZnO or ZnO NPs were added as a source of Zn. It is to be noted that there was no significant difference between the rates of germination when bulk ZnO or nano ZnO was used. This may be due to the soft skin of mung bean seed. The higher dose of TiO₂ NPs decreased the rate of germination. A decrease of 14 and 57% was observed with 200 and 400 mg/kg of soil, respectively. Contrarily, an 8.5% increase in germination rate was observed with 100 mg/Kg of TiO₂. A decrease of 42.8 and 57.1% with 200 and 400 mg/kg soil of Ag NPs, respectively. In the presence of ZSB bacteria almost the same pattern was observed, with the exception that in the presence of ZSB the highest dose of TiO₂ NPs (400) decreased the rate of germination by only 14% (Table 2). This suggests that ZSB conferred tolerance to TiO₂ NPs imposition.

3.4.2 Change in vegetative growth: The data on vegetative growth convincingly shows that the presence of ZSB conferred tolerance to NPs imposition. While the low dose of the NPs (100 mg Kg^− 1, soil) was stimulatory to plant growth (Table 2). Ag NPs at higher dose were more toxic than TiO₂ NPs, and due to the presence of ZSB and ZnO NPs a marked improvement of up to > 30% in pant growth parameters was observed compared to uninoculated control (Table 2, Fig. 4). A decrease in shoot length of 7 and 36% was observed with 200 and 400 mg TiO₂ kg^− 1 of soil without ZSB. While a decrease of only 3 and 21% with the same dose of TiO₂ NPs was observed in the presence of ZSB and ZnO NPs, which shows an improvement of 15% at a dose of 400 mg TiO₂ kg^− 1 of soil. Similarly, with Ag NPs a decrease in shoot length of 9 and 37.5% at a dose of 200 and 400 mg Kg^− 1, respectively was observed without ZSB. While in the presence of ZSB a decrease of 7 and 24% was observed at the same doses. A maximum decrease in root length of 53% was observed with 400 mg Kg^− 1 of TiO₂ NPs without ZSB and ZnO NPs, while in the presence of ZSB at the same dose only a decrease of 22% was observed, showing an improvement of 31%. An improvement of 17% was observed in fresh weight in the presence of ZSB and 400 mg Kg^− 1 of TiO₂ NPs. At the same dose an improvement of 30% in dry weight was observed in the presence of ZSB and ZnO NPs.

In the presence of 400 mg Kg^− 1 Ag NPs a decrease in shoot length, root length, fresh weight, and dry weight of 37, 57, 57 and 53% respectively was observed. While in the presence of ZSB and ZnO NPs, a decrease of 24, 44, 42, and 27% was observed in shoot length, root length, fresh weight, and dry weight, respectively with the same dose of Ag NPs. This shows an improvement of a maximum 26% in dry weight, indicating to the fact that improvement in the presence of Ag NPs were less in compare to TiO₂ NPs. Hence, the Ag NPs were more inhibitory to plant growth than TiO₂ NPs even in the presence of ZSB and ZnO NPs (Table 2, Fig. 4).

3.4.3 Change in pigment and plant Zn content: The change in photosynthetic pigments chlorophyll (a, and b) and carotenoid following treatment with various doses of Ag and TiO₂ NPs in the presence and absence of ZSB and ZnO NPs as source of Zn. It was observed that presence of nanomaterials especially the lower doses (50 and 100 mg kg^− 1 of soil) increased the chlorophyll content in the plant tissues, and a decrease of 6.68 and 10.4% was observed with the highest dose of Ag NPs (400 mg kg^− 1 of soil), without and with ZSB respectively. It is also important to note that the presence of ZSB did not influence the chlorophyll content (Table 2, Fig. 5). The influence of Ag NPs on chlorophyll content was more notable than that of TiO₂ NPs. The carotenoid content increased remarkably with all the test doses and no decrease in carotenoid content was observed. Though this increase slightly plateaued at the highest test dose 400 mg kg-¹ of soil.

The most important aspect of this study was the tissue Zn content which increased at lower doses of 50 and 100 mg NPs kg^− 1 of soil. While, Ag NPs were more inhibitory to Zn assimilation than TiO₂ NPs as even at a dose of 100 mg NPs kg^− 1 of soil a decrease of 11% in Zn content was observed without the ZSB and ZnO NPs (Table 2, Fig. 5). While, at the same dose in the presence of ZSB and ZnO NPs an increase of 6.43% in Zn content was observed. At a high dose of 400 mg NPs kg^− 1 of soil, a decrease in Zn content of 46.3% was observed without ZSB and ZnO NPs. While in the presence of ZSB and ZnO NPs a decrease of 28.8% was observed, showing an improvement of ~ 17% in Zn content. It is puzzling to observe that with TiO₂ NPs the presence of ZSB was not favorable for Zn assimilation as at highest test dose of 400 mg NPs kg^− 1 of soil a decrease in Zn content of 7.6% was observed in the absence of ZSB. While with ZSB a decrease of 32.2% was observed at the same test dose.

Zn is among the most important micronutrient required by all the living organisms, ensuring its adequate supply to plants will improve plant growth, yield of agricultural products, and their Zn status. Provision of Zn dense diet will improve the Zn status in human that will translate in various health benefits, reduced health care cost and will improve overall productivity (Khan and Khan, 2022; Khan et al., 2022b). Agronomic Zn biofortification employing ZSBs is one of the best, economic and feasible approach to address the problem of Zn deficiency (Saleem and Khan, 2022; Saleem et al., 2023b, a). With tons of engineered nanomaterials released in soil, ENMs are emerging as new environmental pollutant having obvious consequences on soil microbiome and processes mediated by soil bacteria (Khan et al., 2022a). Zn solubilization by bacteria is one such important process which plays a significant role in improving Zn bioavailability to plants (Haroon et al., 2022). In soils already burdened with Zn deficiency how the presence of ENMs influences the activity of ZSB and what are the consequences on the vegetative growth of Mung bean? was investigated. Two strains of bacteria namely Bacillus sp. D-7 and P. aeruginosa, D-117 exhibiting good Zn solubilization efficiency were used for the study. Zn solubilization by various bacteria including Bacillus and Pseudomonas has been reported earlier also (Kamran et al., 2017; Costerousse et al., 2018; Saleem and Khan, 2022). But we chose the strains not only based on their Zn solubilization potential but also based on the presence of additional multiple PGP traits. Use of such strains will improve Zn status of the plant and will also promote plant growth through additional mechanisms like production of IAA, siderophore, ammonia and HCN. Our results show that compared to uninoculated control, plant growth parameters improved in the presence of these strains even without ZnO NPs as source of Zn, confirming that the presence of these strains trigger a plant growth response through other mechanisms also. While the addition of ZnO NPs as source of Zn in addition to ZSB further improved the vegetative growth of Mung bean compared to control without Zn or bulk Zn (Table 2). ZnO NPs are known to promote plant growth as a better source of Zn than the bulk ZnO for Mung bean in this study. We also have reported earlier the same for wheat (Singh et al., 2019; Mazhar et al., 2023; Saleem et al., 2023b). But what happens if other ENMs like Ag NPs or TiO₂ NPs are added as pollutant to soil?

It was found that the lower doses of both Ag and TiO₂ NPs (50, 100 mg kg^− 1 of NPs), trigger plant growth response in Mung bean. This growth stimulation may be due to various reasons. Nano TiO₂ is found to improve photosynthesis, and activities of rubisco and antioxidant enzymes (Lei et al., 2008; Lyu et al., 2017). In addition to this an increase in phenolics and flavonoid compounds following treatment with TiO₂-NPs is also reported (Mohajjel Shoja et al., 2021). Improvement of these plant physiological processes result in improved plant growth. Another important reason of ENMs mediated growth promotion may be the influence of ENMs on multiple plant-microbe interactions. Very important though, the interplay of plant-microbe interactions is often over looked while evaluating toxicity of pollutants. As an example, we earlier found that ZnO NPs trigger the production of pyrrolnitrin by Pseudomonas aeruginosa PAO1 which increases its antifungal activity providing protection to plants against fungal pathogens (Khan et al., 2018). In another recent culture-based study on Zn biofortification of wheat we observed that lower dose of ZnO NPs, stimulated the populations of phosphate solubilizing bacteria and nitrogen fixers (Saleem et al., 2023b). The increase in nitrogen fixing bacteria may improve the supply of nitrogen to plants which might have also resulted in an increase in the protein content of wheat grains as observed in our study (Saleem et al., 2023b). The same change in the microbial community was confirmed using next generation sequencing analysis of soil microbial community following amendment with ZnO-NPs (Saleem et al., 2023b).

But this growth promoting response is generally observed at low doses and higher doses (200 and 400 mg NPs Kg^− 1 of soil) were found to inhibit plant growth as evident from decrease in shoot length, root length, fresh weight, and dry weight (Table 2). The toxicity of nanomaterials varies with their concentration, size, shape, and many other factors (Khan et al., 2015). The toxicity of nano TiO₂ at higher doses to plants has been reported in many studies (Ghosh et al., 2010; Cox et al., 2016; Ameen et al., 2021). Various mechanisms for the nano TiO₂ toxicity to plants have been proposed and demonstrated, including DNA damage (Ghosh et al., 2010). It is surprising that the chlorophyll content did not decrease even in the presence of higher doses of TiO₂ NPs. The rise of chlorophyll content was comparatively less significant than carotenoid. Many studies have documented an increase in Chlorophyll content and antioxidant activity of plants following treatment with ENMs. Interestingly in the presence of ZSB the decrease in plant growth plateaued even by 30% in some cases, proving that the presence of ZnO NPs and ZSB help in mitigating the toxicity of the ENMs (Table 2). There can be various reasons for this increased resilience of Mung bean to ENMS in the presence of ZSB and ZnO NPs. The increased bioavailability of Zn to plants increases the vegetative growth in plants and thus protect plants from the toxicity of other metals (Dhaliwal et al., 2023). It has been demonstrated that ZnO-NPs increase the antioxidative capacity of tomato plants and trigger an increased production of flavonoids and alkaloids which may help in mitigating Cadmium toxicity (Sun et al., 2023). Presence of ZnO NPs may also minimize toxicity through competitive binding to metal receptors in plants decreasing the uptake consequently reducing the toxicity of other metals (Angulo-Bejarano et al., 2021).

In the presence of Ag NPs also, similar results were obtained, lower doses promoted plant growth while higher doses of Ag NPs decreased plant growth. Though the reduction in vegetative growth was greater with Ag NPs than with TiO₂ NPs (Table 2, Fig. 4). A number of studies document higher toxicity of Ag NPs on plants (Song et al., 2013). The mechanisms of Ag NPs toxicity to plant includes generation of excess ROS species, lipid peroxidation, inhibition of electron transport chain and DNA damage (Nair et al., 2010; Patlolla et al., 2012; Nair and Chung, 2014; Cox et al., 2016; Küpper and Andresen, 2016). Moreover, it is important to note that in the presence of Ag NPs improvement of vegetative growth parameters with ZSB and ZnO NPs was less than what was observed for TiO₂ NPs. Which further confirms the higher toxicity of Ag NPs. Most importantly the presence of ZSB helped in maintaining Zn density in plant tissues even in the presence of higher doses of TiO₂ NPs or Ag NPs. This study therefore determines the threshold levels of the two nanomaterials that can be used in field without affecting crop if needed and confirms that the use of ZSB helps in mitigating the toxicity of TiO₂ and Ag NPs and in improving the Zn status of the crops. Detailed studies are required to understand the mechanism underlying the mitigation of ENMs toxicity.

This study confirms that Zinc solubilizing bacteria can be successfully used for improving vegetative growth and the Zn status of Mung bean. The presence of low doses of TiO₂ and Ag NPs improve vegetative growth of the plant, but the higher doses of the two ENMs decreased the vegetative growth. Chlorophyll and carotenoid content did not decrease even at the higher doses of ENMs. The use of ZSB and ZnO NPs helped in mitigating the toxicity of the two ENMs, and in maintaining the Zn status of the crop under nanomaterial stress. Future detailed studies are required to clearly understand the mechanism through which the ZSB and ZnO NPs mitigate the toxicity of TiO₂ and Ag NPs.

Data availability

Data is submitted to GenBank, National Center for Biotechnology Information (NCBI Bethesda MD, USA) and accession numbers are included in the manuscript.

Declaration of Competing Interest

The authors declare that they have no conflict of interests what so ever.

Authors Contribution

Mahvash Haroon: Experiments, data collection, analysis. Shams Tabrez Khan: Project management, Formulation of study and Preparation of manuscript. Abdul Malik: Advisory and Guidance

Acknowledgement

Authors acknowledge the generous funding by MHRD Govt. of India through SPARC project P594.

Ahmed, M.A., Shafiei-Masouleh, S.-S., Mohsin, R.M., Salih, Z.K., 2023. Foliar Application of Iron Oxide Nanoparticles Promotes Growth, Mineral Contents, and Medicinal Qualities of Solidago virgaurea L. J. Soil Sci. Plant Nutr. 23, 2610-2624.
Alloway, B.J., 2008. Zinc in Soils and Crop Nutrition. International Zinc Association.
Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl. Acids Res. 25, 3389-3402.
Ameen, F., Alsamhary, K., Alabdullatif, J.A., ALNadhari, S.J.E., Safety, E., 2021. A review on metal-based nanoparticles and their toxicity to beneficial soil bacteria and fungi. 213, 112027.
Angulo-Bejarano, P.I., Puente-Rivera, J., Cruz-Ortega, R., 2021. Metal and Metalloid Toxicity in Plants: An Overview on Molecular Aspects. Plants 10, 635.
Arnon, D.I., 1949. Copper enzymes in isolated chloroplasts polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1-15.
Barrow, G.I., Feltham, R.K.A., 1993. Cowan and Steel's Manual for the Identification of Medical Bacteria, 3 ed. Cambridge University Press, Cambridge.
Berendsen, R.L., Pieterse, C.M.J., Bakker, P.A.H.M., 2012. The rhizosphere microbiome and plant health. Trends Plant Sci. 17, 478-486.
Cappuccino, J., Sherman, N.J.M.A.L.M., 3rd ed., Benjamin/Cummings Pub Co.: Redwood City, C., USA, 1992. Ammonia production. 125-179.
Cappuccino, J.G., Sherman, N., 1999. Microbiology: A Laboratory Manual. Benjamin/Cummings.
Costerousse, B., Schönholzer-Mauclaire, L., Frossard, E., Thonar, C., 2018. Identification of Heterotrophic Zinc Mobilization Processes among Bacterial Strains Isolated from Wheat Rhizosphere (Triticum aestivum L.). Appl. Environ. Microbiol. 84, e01715-01717.
Cox, A., Venkatachalam, P., Sahi, S., Sharma, N., 2016. Silver and titanium dioxide nanoparticle toxicity in plants: A review of current research. Plant physiology and biochemistry : PPB 107, 147-163.
Dhaliwal, S.S., Sharma, V., Shukla, A.K., Kaur, M., Kaur, J., Verma, V., Singh, P., Barek, V., Gaber, A., Hossain, A., 2023. Biofortification of mungbean (Vigna radiata L. (Wilczek)) with boron, zinc and iron alters its grain yield and nutrition. Sci. Rep. 13, 3506.
Donate-Correa, J., León-Barrios, M., Pérez-Galdona, R., 2005. Screening for plant growth-promoting rhizobacteria in Chamaecytisus proliferus (tagasaste), a forage tree-shrub legume endemic to the Canary Islands. Plant and Soil 266, 261-272.
Faizan, M., Faraz, A., Yusuf, M., Khan, S.T., Hayat, S., 2018. Zinc oxide nanoparticle-mediated changes in photosynthetic efficiency and antioxidant system of tomato plants. Photosynthetica 56, 678-686.
Fasim, F., Ahmed, N., Parsons, R., Gadd, G.M., 2002. Solubilization of zinc salts by a bacterium isolated from the air environment of a tannery. FEMS Microbiol. Lett. 213, 1-6.
Ghosh, M., Bandyopadhyay, M., Mukherjee, A., 2010. Genotoxicity of titanium dioxide (TiO₂) nanoparticles at two trophic levels: Plant and human lymphocytes. Chemosphere 81, 1253-1262.
Gordon, S.A., Weber, R.P., 1951. Colorimetric estimation of indole acetic acid. Plant. Physiol. 26, 192-195.
Haddad, L., Hawkes, C., Webb, P., Thomas, S., Beddington, J., Waage, J., Flynn, D., 2016. A new global research agenda for food. Nature 540, 30-32.
Haroon, M., Khan, S. T., & Malik, A. (2022). Zinc-solubilizing bacteria: an option to increase zinc uptake by plants. in: Khan, S.T., Malik, A. (Eds.). Microbial Biofertilizers and Micronutrient Availability: The Role of Zinc in Agriculture and Human Health. Springer International Publishing, Cham, pp. 207-238
Hols, P., Ferain, T., Garmyn, D., Bernard, N., Delcour, J., 1994. Use of homologous expression-secretion signals and vector-free stable chromosomal integration in engineering of Lactobacillus plantarum for alpha-amylase and levanase expression. Appl. Environ. Microbiol. 60(5): 1401–1413.
Huang, X., Madan, A., 1999. CAP3: A DNA sequence assembly program. Genome. Res. 9, 868-877.
Kamran, S., Shahid, I., Baig, D.N., Rizwan, M., Malik, K.A., Mehnaz, S., 2017. Contribution of Zinc Solubilizing Bacteria in Growth Promotion and Zinc Content of Wheat. Front. Microbiol. 8, 2593-2593.
Kang, M.e., Weng, Y., Liu, Y., Wang, H., Ye, L., Gu, Y., Bai, X., 2023. A Review on the Toxicity Mechanisms and Potential Risks of Engineered Nanoparticles to Plants. Rev. Environ. Contam. Toxicol. 261, 5.
Keller, A.A., McFerran, S., Lazareva, A., Suh, S., 2013. Global life cycle releases of engineered nanomaterials. J. Nanopart Res. 15, 1692.
Khan, S., Al-Khedhairy, A., Musarrat, J., 2015. ZnO and TiO₂ nanoparticles as novel antimicrobial agents for oral hygiene: a review. J. Nanopart Res. 17.
Khan, S.T., 2020. Interaction of Engineered Nanomaterials with Soil Microbiome and Plants: Their Impact on Plant and Soil Health. in: Hayat, S., Pichtel, J., Faizan, M., Fariduddin, Q. (Eds.). Sustainable Agriculture Reviews 41: Nanotechnology for Plant Growth and Development. Springer International Publishing, Cham, pp. 181-199.
Khan, S.T., 2022. Consortia-based microbial inoculants for sustaining agricultural activities. Appl. Soil Ecol. 176, 104503.
Khan, S.T., Adil, S.F., Shaik, M.R., Alkhathlan, H.Z., Khan, M., Khan, M., 2022a. Engineered Nanomaterials in Soil: Their Impact on Soil Microbiome and Plant Health. Plants, 11, 109.
Khan, S.T., Ahmad, J., Ahamed, M., Jousset, A., 2018. Sub-lethal doses of widespread nanoparticles promote antifungal activity in Pseudomonas protegens CHA0. Sci. Total Environ. 627, 658-662.
Khan, S.T., Khan, M.A., 2022. Strategies to Counter Zinc Deficiency, Current Status and Future Directions. in: Khan, S.T., Malik, A. (Eds.). Microbial Biofertilizers and Micronutrient Availability: The Role of Zinc in Agriculture and Human Health. Springer International Publishing, Cham, pp. 431-456.
Khan, S.T., Malik, A., 2019. Engineered nanomaterials for water decontamination and purification: From lab to products. J. Hazard. Mater. 363, 295-308.
Khan, S.T., Malik, A., Alwarthan, A., Shaik, M.R., 2022b. The enormity of the zinc deficiency problem and available solutions; an overview. Arab. J. Chem. 15, 103668.
Khan, S.T., Musarrat, J., Al-Khedhairy, A.A., 2016. Countering drug resistance, infectious diseases, and sepsis using metal and metal oxides nanoparticles: Current status. Coll. Surf. B, Biointerfaces 146, 70-83.
Khan, S.T., Nakagawa, Y., Harayama, S., 2007. Galbibacter mesophilus gen. nov., sp. nov., a novel member of the family Flavobacteriaceae. Int. J. Syst. Evol. Microbiol. 57, 969-973.
Küpper, H., Andresen, E., 2016. Mechanisms of metal toxicity in plants. Metallomics 8, 269-285.
Lei, Z., Mingyu, S., Xiao, W., Chao, L., Chunxiang, Q., Liang, C., Hao, H., Xiaoqing, L., Fashui, H., 2008. Antioxidant stress is promoted by nano-anatase in spinach chloroplasts under UV-B radiation. Biol. Trace Elem. Res. 121, 69-79.
Lichtenthaler, H.K., Wellburn, A.R., 1983. Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochem. Soc. Transac. 11: 591-592.
Lindsay, W.L., Norvell, W.A., 1978. Development of a DTPA soil test for Zinc, Iron, Manganese, and Copper. Soil Sci. Soc. Amer. J. 42, 421-428.
Lorck, H., 1948. Production of Hydrocyanic Acid by Bacteria. Physiol. Plant. 1, 142-146.
Lyu, S., Wei, X., Chen, J., Wang, C., Wang, X., Pan, D., 2017. Titanium as a Beneficial Element for Crop Production. Front. Plant. Sci. 8. https://doi.org/10.3389/fpls.2017.00597
Mazhar, Z., Akhtar, J., Alhodaib, A., Naz, T., Zafar, M.I., Iqbal, M.M., Fatima, H., Naz, I., 2023. Efficacy of ZnO nanoparticles in Zn fortification and partitioning of wheat and rice grains under salt stress. Sci. Rep. 13, 2022.
Mohajjel Shoja, H., Ahmadi, L., Kolahi, M., Mohajel Kazemi, E., 2021. Effect of TiO₂ NPs on the growth, anatomic features and biochemistry parameters of Baby sun rose (Aptenia cordifolia). Physiol. Mol. Biol. Plants. 27, 2071-2081.
Nair, P.M.G., Chung, I.M., 2014. Physiological and molecular level studies on the toxicity of silver nanoparticles in germinating seedlings of mung bean (Vigna radiata L.). Acta Physiol. Plant. 37, 1719.
Nair, R., Varghese, S.H., Nair, B.G., Maekawa, T., Yoshida, Y., Kumar, D.S., 2010. Nanoparticulate material delivery to plants. Plant. Sci. 179, 154-163.
Nautiyal, C.S., 1999. An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiol. Lett. 170, 265-270.
Patlolla, A.K., Berry, A., May, L., Tchounwou, P.B. 2012. Genotoxicity of silver nanoparticles in Vicia faba: a pilot study on the environmental monitoring of nanoparticles. Int. J. Environ. Res. Pub. Health. 9(5): 1649–1662.
Patten, C.L., Glick, B.R., 1996. Bacterial biosynthesis of indole-3-acetic acid. Can. J. Microbiol. 42, 207-220.
Piccinno, F., Gottschalk, F., Seeger, S., Nowack, B., 2012. Industrial production quantities and uses of ten engineered nanomaterials in Europe and the world. J. Nanopart. Res. 14, 1109.
Poppy, G.M., Baverstock-Poppy, J.J., Baverstock, J., 2022. Trade and dietary preferences can determine micronutrient security in the United Kingdom. Nature Food 3, 512-522.
Rathje, 1959. Jackson, M. L.: Soil chemical analysis. Verlag: Prentice Hall, Inc., Englewood Cliffs, NJ. 1958, 498 S. DM 39.40. 85, 251-252.
Saleem, S., Khan, S.T., 2022. Development of Microbes-Based Biofertilizer for Zinc Dissolution in Soil. in: Khan, S.T., Malik, A. (Eds.). Microbial Biofertilizers and Micronutrient Availability: The Role of Zinc in Agriculture and Human Health. Springer International Publishing, Cham, pp. 299-329.
Saleem, S., Malik, A., Khan, S.T., 2023a. Prospects of ZnO-Nanoparticles for Zn Biofortification of Triticum aestivum in a Zinc-Solubilizing Bacteria Environment. J. Soil Sci. Plant Nutr. 23, 4350-4360.
Saleem, S., Malik, A., Khan, S.T., 2023b. ZnO nanoparticles in combination with Zn biofertilizer improve wheat plant growth and grain Zn content without significantly changing the rhizospheric microbiome. Environ. Exp. Bot. 213, 105446.
Salehi, H., Cheheregani Rad, A., Raza, A., Djalovic, I., Prasad, P.V.V., 2023. The comparative effects of manganese nanoparticles and their counterparts (bulk and ionic) in Artemisia annua plants via seed priming and foliar application. Front. Plant Sci. 13. https://doi.org/10.3389/fpls.2022.1098772
Schwyn, B., Neilands, J.B., 1987. Universal chemical assay for the detection and determination of siderophores. Anal. Biochem. 160, 47-56.
Singh, J., Kumar, S., Alok, A., Upadhyay, S.K., Rawat, M., Tsang, D.C.W., Bolan, N., Kim, K.-H., 2019. The potential of green synthesized zinc oxide nanoparticles as nutrient source for plant growth. J. Clean. Prod. 214, 1061-1070.
Song, U., Jun, H., Waldman, B., Roh, J., Kim, Y., Yi, J., Lee, E.J., 2013. Functional analyses of nanoparticle toxicity: A comparative study of the effects of TiO₂ and Ag on tomatoes (Lycopersicon esculentum). Ecotoxicol. Environ. Saf. 93, 60-67.
Sun, L., Wang, R., Ju, Q., Xing, M., Li, R., Li, W., Li, W., Wang, W., Deng, Y., Xu, J., 2023. Mitigation mechanism of zinc oxide nanoparticles on cadmium toxicity in tomato. Front. Plant Sci. 14: 1162372. doi: 10.3389/fpls.2023.1162372

Table 1. Quantitative and qualitative Zinc solubilizing potential of the isolates determined on basal medium containing either ZnO or ZnCO₃ as insoluble Zn and in liquid medium, respectively. Solubilization efficiency (SE) was calculated on solid medium as described earlier (Saleem et al 2023).

S. No.	Strain	Qualitative Assay				Quantitative Assay
		Zn Solubilization Zone (Diameter in mm )		Zn Solubilization Efficiency ZSE (%)		Zn solubilized (mg/ml)
		ZnO	ZnCO₃	ZnO	ZnCO₃	ZnO	ZnCO₃
1	D-7	30 ± 0.14	28 ± 0.19	272.73	254.55	2.26 ± 0.43	1.78 ± 0.23
2	D-8	29 ± 0.17	28 ± 0.23	263.64	254.54	1.90 ± 0.59	0.89 ± 0.41
3	D-9	27 ± 0.15	28 ± 0.15	245.45	254.55	1.57 ± 0.42	1.53 ± 0.34
4	D-23	28 ± 0.21	30 ± 0.18	254.55	272.73	2.09 ± 0.61	2.41 ± 0.28
5	D-32	27 ± 0.19	25 ± 0.27	245.45	227.27	1.50 ± 0.32	1.45 ± 0.37
6	D-33	27 ± 0.11	25 ± 0.16	254.71	240.38	1.60 ± 0.47	1.36 ± 0.29
7	D-44	28 ± 0.17	24 ± 0.11	254.54	218.18	1.48 ± 0.63	1.24 ± 0.38
8	D-45	29 ± 0.13	24 ± 0.18	263.63	218.18	1.56 ± 0.71	1.35 ± 0.35
9	D-62	27 ± 0.26	15 ± 0.19	245.45	136.36	1.59 ± 0.29	1.35 ± 45
10	D-69	30 ± 0.23	17 ± 0.22	272.73	154.54	1.78 ± 0.48	1.21 ± 0.51
11	D-72	30 ± 0.18	26 ± 0.24	272.72	236.36	1.87 ± 0.79	1.91 ± 0.21
12	D-76	28 ± 0.14	11 ± 0.17	254.55	115.78	1.30 ± 0.75	1.13 ± 0.26
13	D-88	28 ± 0.22	26 ± 0.15	254.55	236.36	2.10 ± 0.37	1.12 ± 0.38
14	D-106	27 ± 0.25	16 ± 0.21	245.45	145.45	1.99 ± 0.49	1.39 ± 0.32
15	D-117	28 ± 0.18	26 ± 0.19	280.00	260.00	2.31 ± 0.54	1.97 ± 0.21
16	D-7-20	28 ± 0.11	28 ± 0.15	254.55	254.54	1.93 ± 0.38	1.39 ± 0.27

Table 2. Changes in rate of germination, shoot length, root length, fresh and dry weights per plant with various doses of soil abundant nanoparticles (TiO2 and Ag) in the presence of ZnO NPs and Zn biofertililzer with multiple plant growth promoting traits.

Treatment	Germination	Shoot Length (cm/plant)	Root Length (cm/plant)	Fresh Weight (g/plant)	Dry Weight (g/plant)	Total Chlorophyll	Carotenoid	Tissue Zinc (ppm)
Cont	2.8 ± 0.71	17.47 ± 0.43	3.42 ± 0.25	3.55 ± 0.26	2.27 ± 0.12	10.12 ± 1.30	1.46 ± 0.148	16.30 ± 0.85
ZB	3.0 ± 0.71	21.565 ± 0.05	3.75 ± 0.08	5.15 ± 0.39	2.72 ± 0.17	11.01 ± 0.22	1.54 ± 0.289	17.00 ± 0.91
B_ZB	3.5 ± 0.32	22.2 ± 0.32	4.06 ± 0.05	5.82 ± 0.22	3.02 ± 0.09	11.15 ± 0.80	1.79 ± 0.028	20.11 ± 1.11
ZN	3.5 ± 0.22	23.44 ± 0.25	4.36 ± 0.04	6.42 ± 0.06	3.31 ± 0.06	12.12 ± 0.69	1.63 ± 0.431	23.5 ± 1.23
ZN_T50	3.5 ± 0.71	24.23 ± 0.3	5.16 ± 0.21	7.25 ± 0.13	3.35 ± 0.19	14.21 ± 0.84	3.64 ± 0.544	43.7 ± 1.85
ZN_T100	3.8 ± 0.54	24.87 ± 0.25	5.28 ± 0.16	7.37 ± 0.21	3.93 ± 0.15	14.67 ± 0.41	3.99 ± 0.106	37.4 ± 1.73
ZN_T200	3.0 ± 0.32	21.73 ± 0.32	3.88 ± 0.19	4.97 ± 0.40	2.20 ± 0.26	13.4 ± 0.43	3.4 ± 0.042	29.1 ± 1.55
ZN_T400	1.5 ± 0.70	15.02 ± 0.32	2.05 ± 0.21	3.05 ± 0.21	1.85 ± 0.07	11.95 ± 1.69	3.00 ± 0.424	21.7 ± 1.08
B_ZN	3.5 ± 0.31	24.21 ± 0.19	4.96 ± 0.10	6.65 ± 0.20	3.31 ± 0.08	12.88 ± 0.97	2.02 ± 0.049	26.4 ± 1.32
B_ZN_T50	3.5 ± 0.51	24.99 ± 0.3	5.55 ± 0.08	7.12 ± 0.17	3.75 ± 0.13	14.82 ± 0.88	3.93 ± 0.487	31.6 ± 1.55
B_ZN_T100	4.0 ± 0.43	25.77 ± 0.15	5.72 ± 0.10	8.03 ± 0.15	4.80 ± 0.10	15.18 ± 0.58	4.12 ± 0.162	29.7 ± 1.38
B_ZN_T200	3.8 ± 0.28	23.43 ± 0.5	4.33 ± 0.15	6.13 ± 0.32	3.17 ± 0.06	13.78 ± 0.09	3.51 ± 0.226	24.5 ± 1.15
B_ZN_T400	3.0 ± 0.39	19.23 ± 0.18	3.85 ± 0.07	4.31 ± 0.16	2.85 ± 0.07	13.45 ± 1.29	3.49 ± 0.395	17.9 ± 0.95
ZN	3.5 ± 0.22	23.44 ± 0.25	4.36 ± 0.04	6.42 ± 0.06	3.31 ± 0.06	12.12 ± 0.69	1.63 ± 0.431	23.5 ± 1.23
ZN_A50	3.5 ± 0.68	23.80 ± 0.22	4.91 ± 0.22	6.62 ± 0.35	3.32 ± 0.26	13.85 ± 0.77	3.27 ± 0.622	24.9 ± 1.42
ZN_A100	3.0 ± 0.58	24.42 ± 0.33	4.92 ± 0.07	6.77 ± 0.21	3.97 ± 0.30	14.15 ± 0.31	3.62 ± 0.035	20.9 ± 1.05
ZN_A200	2.0 ± 0.12	21.30 ± 0.07	3.52 ± 0.19	4.6 ± 0.14	2.40 ± 0.10	13.16 ± 0.56	3.23 ± 0.388	15.4 ± 1.00
ZN_A400	1.5 ± 0.09	14.65 ± 0.21	1.87 ± 0.11	2.75 ± 0.07	1.55 ± 0.07	11.31 ± 1.68	2.08 ± 0.749	12.6 ± 1.51
B_ZN	3.5 ± 0.31	24.21 ± 0.19	4.96 ± 0.10	6.65 ± 0.20	3.31 ± 0.08	12.88 ± 0.97	2.02 ± 0.049	26.4 ± 1.32
B_ZN_A50	3.5 ± 0.63	24.45 ± 0.13	5.34 ± 0.09	6.68 ± 0.17	3.75 ± 0.13	14.29 ± 0.49	3.57 ± 0.247	29.3 ± 1.12
B_ZN_A100	3.5 ± 0.71	25.28 ± 0.1	5.41 ± 0.03	7.43 ± 0.06	4.67 ± 0.25	14.13 ± 0.48	3.44 ± 0.042	28.1 ± 1.25
B_ZN_A200	2 ± 0.28	22.47 ± 0.27	4.04 ± 0.10	5.7 ± 0.10	2.80 ± 0.10	13.42 ± 0.89	3.37 ± 0.141	23.4 ± 1.32
B_ZN_A400	1.5 ± 0.13	18.45 ± 0.5	2.78 ± 0.12	3.88 ± 0.04	2.40 ± 0.07	11.54 ± 1.35	3.09 ± 0.579	18.8 ± 1.11

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A combination of Zinc solubilizing bacteria and ZnO nanoparticles alleviates the toxicity of Ag and TiO₂ nanoparticles in Mung bean

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 Characterization of nanoparticles

2.2 Soil sampling and isolation of aerobic heterotrophs

2.3 Zinc Solubilization assay

2.4 In vitro screening for PGP traits

2.5 Polyphasic characterization of ZSB

2.6 Activity of ZSB in an environment of Ag and TiO₂ nanoparticles and Mung bean rhizosphere

2.8 Zn concentration in soil and plant tissues

3. Results

3.1 Characterization of nanoparticles

3.2 Isolation of bacteria screening for Zn solubilization potential and PGP traits

3.3 Polyphasic characterization of efficient ZSB strains

3.4 Activity of ZSB in an environment of Ag and TiO₂ NPs in Mung bean rhizosphere

4. Discussion

5. Conclusion

Declarations

References

Tables

Supplementary Tables

Additional Declarations

Status:

Version 1

A combination of Zinc solubilizing bacteria and ZnO nanoparticles alleviates the toxicity of Ag and TiO2 nanoparticles in Mung bean

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 Characterization of nanoparticles

2.2 Soil sampling and isolation of aerobic heterotrophs

2.3 Zinc Solubilization assay

2.4 In vitro screening for PGP traits

2.5 Polyphasic characterization of ZSB

2.6 Activity of ZSB in an environment of Ag and TiO2 nanoparticles and Mung bean rhizosphere

2.8 Zn concentration in soil and plant tissues

3. Results

3.1 Characterization of nanoparticles

3.2 Isolation of bacteria screening for Zn solubilization potential and PGP traits

3.3 Polyphasic characterization of efficient ZSB strains

3.4 Activity of ZSB in an environment of Ag and TiO2 NPs in Mung bean rhizosphere

4. Discussion

5. Conclusion

Declarations

References

Tables

Supplementary Tables

Additional Declarations

Status:

Version 1

A combination of Zinc solubilizing bacteria and ZnO nanoparticles alleviates the toxicity of Ag and TiO₂ nanoparticles in Mung bean

2.6 Activity of ZSB in an environment of Ag and TiO₂ nanoparticles and Mung bean rhizosphere

3.4 Activity of ZSB in an environment of Ag and TiO₂ NPs in Mung bean rhizosphere